Methods for treating progressive cognitive disorders related to neurofibrillary tangles

ABSTRACT

The described invention provides methods for treating or preventing progression of a progressive cognitive disease, disorder or condition, and methods for improving resilience of cognitive function in a subject in need thereof.

This application is a divisional application of U.S. patent applicationSer. No. 12/470,427, filed May 21, 2009, which claims the benefit ofpriority of U.S. application 61/055,009, filed May 21, 2008,incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with government support under Grant NumberSBIR-1R43AG029670 awarded by the National Institute on Aging. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The described invention relates to methods for treating a progressivecognitive disorder and methods for improving resilience of cognitivefunction.

BACKGROUND Alzheimer's Disease

Alzheimer's disease (also called “AD”, “senile dementia of the AlzheimerType (SDAT)” or “Alzheimer's”) is a neurodegenerative disorder of thecentral nervous system (“CNS”). AD is usually diagnosed clinically fromthe patient history, collateral history from relatives, and clinicalobservations, based on the presence of characteristic neurological andneuropsychological features.

AD is characterized by loss of neurons and synapses in the cerebralcortex and certain subcortical regions. This loss results in grossatrophy of the affected regions, including degeneration in the temporallobe and parietal lobe, and parts of the frontal cortex and cingulategyms. Both amyloid plaques (“AP”) and neurofibrillary tangles (“NFT”)are clearly visible by microscopy in brains of those afflicted with AD.Plaques are dense, mostly insoluble deposits of amyloid-beta (“Aβ”)protein and cellular material outside and around neurons. NFT areaggregates of the microtubule-associated protein “tau”, which havebecome hyperphosphorylated and accumulate inside the cells themselves.Although many older individuals develop some plaques and tangles as aconsequence of ageing, the brains of AD patients have a greater numberof such plaques and tangles in specific brain regions, such as thetemporal lobe.

AD is characterized histologically by the presence of extracellularamyloid deposits in the brain, together with widespread neuronal loss.Extracellular amyloid deposits are known as neuritic or senile plaques.Amyloid deposits also may be found within and around blood vessels. Themain protein constituent of AD and AD-like senile plaques is Aβ. Aβ maybe detected in plasma and cerebrospinal fluid (“CSF”) in vivo, and incell culture media in vitro.

The terms “amyloid peptide” “amyloid β peptide” and “Aβ” are usedinterchangeably herein to refer to the family of peptides generatedthrough proteolytic processing of the amyloid precursor protein (APP).

APP exists as three different spliced isoforms, one having 770 aminoacids (isoform a) (SEQ ID NO:1), one having 751 amino acids (isoform b)(SEQ ID NO:2), and one having 695 amino acids (SEQ ID NO:3). The term“APP” as used herein refers to all three isoforms. The terms “amyloidpeptide” “amyloid β peptide” and “Aβ” include, but are not limited to,Aβ40 (SEQ ID NO:4), Aβ42 (SEQ ID NO:5) and Aβ43 (SEQ ID NO:6). The twomajor forms of Aβ are Aβ40 (SEQ ID NO:4), corresponding to a 40 aminoacid-long peptide and Aβ42 (SEQ ID NO:5), corresponding to a 42 aminoacid-long peptide. Aβ43 (SEQ ID NO:6) corresponds to a 43 aminoacid-long Aβ peptide.

It generally is believed that brain lipids are intricately involved inAβ-related pathogenic pathways. The Aβ peptide is the majorproteinaceous component of the amyloid plaques found in the brains of ADpatients and is regarded by many as the culprit of the disorder. Theamount of extracellular Aβ accrued is critical for the pathobiology ofAD and depends on the antagonizing rates of its production/secretion andits clearance. Studies have shown that neurons depend on the interactionbetween Presenilin 1 (“PS1”) and Cytoplasmic-Linker Protein 170(“CLIP-170”) to both generate Aβ and to take it up through thelipoprotein receptor related protein (“LRP”) pathway. Further to thisrequirement, formation of Aβ depends on the assembly of key proteins inlipid rafts (“LRs”). The term “lipid rafts” as used herein refers tomembrane microdomains enriched in cholesterol, glycosphingolipids andglucosylphosphatidyl-inositol-(GPI)-tagged proteins implicated in signaltransduction, protein trafficking and proteolysis. Within the LRs it isbelieved that Aβ's precursor, Amyloid Precursor Protein (“APP”), a typeI membrane protein, is cleaved first by the protease β-secretase (BACE)to generate the C-terminal intermediate fragment of APP, CAPPβ, whichremains embedded in the membrane. CAPPβ subsequently is cleaved at asite residing within the lipid bilayer by γ-secretase, a high molecularweight multi-protein complex containing presenilin, (PS1/PS2),nicastrin, PEN-2, and APH-1 or fragments thereof. Aβ finally is releasedoutside the cell, where it may: i) start accumulating followingoligomerization and exerting toxicity to neurons, or ii) be removedeither by mechanisms of endocytosis (involving apolipoprotein-E (apoE)and LRP or Scavenger Receptors) or by degradation by extracellularproteases including insulin-degrading enzyme (IDE) and neprilysin.

It generally is believed that soluble Aβ oligomers, prior to plaquebuildup, exert neurotoxic effects leading to neurodegeneration, synapticloss and dementia. Further, increased Aβ levels may result from abnormallipid accumulation, thereby producing altered membrane fluidity andlipid raft composition.

The presence of NFT is a characteristic of AD brains. These aggregationsof hyperphosphorylated tau protein also are referred to as “PairedHelical filaments” (PHF). The role of PHF, whether as a primarycausative factor in AD or in a more peripheral role, is uncertain.However, the accumulation of PHF cause the destabilization of themicrotubule network, thus compromising neuronal scaffolding anddisrupting cellular trafficking and signal transduction/communication,and leading to neuronal death.

NFT are not specific to AD; NFT also are seen in Creutzfeldt-Jakobdisease, Supranuclear Palsy, corticobasal neurodegeneration andFrontaltemporal Dementia with Parkinsonism linked to chromosome 17(FTDP-17). This suggests that NFT may represent endpoints leading toneurodegeneration, which may be generated by a number of causativeevents/insults.

Leptin

Leptin is a helical protein secreted by adipose tissue, which acts on areceptor site in the ventromedial nucleus of the hypothalamus to curbappetite and increase energy expenditure as body fat stores increase.Leptin levels are 40% higher in women, and show a further 50% rise justbefore menarche, later returning to baseline levels. Leptin levels arelowered by fasting and increased by inflammation.

Human genes encoding both leptin and the leptin receptor site have beenidentified. Laboratory mice having mutations on the ob gene, whichencodes leptin, become morbidly obese, diabetic, and infertile;administration of leptin to these mice improves glucose tolerance,increases physical activity, reduces body weight by 30%, and restoresfertility. Mice with mutations of the db gene, which encodes the leptinreceptor, also become obese and diabetic but do not improve withadministration of leptin. Although mutations in both the leptin andleptin receptor genes have been found in a small number of morbidlyobese human subjects with abnormal eating behavior, the majority ofobese persons do not show such mutations, and have normal or elevatedcirculating levels of leptin. The immune deficiency seen in starvationmay result from diminished leptin secretion. Mice lacking the gene forleptin or its receptor show impairment of T-cell function, and, inlaboratory studies, leptin has induced a proliferative response in humanCD4 lymphocytes.

Leptin also controls insulin sensitivity. Within the CNS, leptin crossesthe blood brain barrier to bind specific receptors in the brain tomediate food intake, body weight and energy expenditure. In general, (i)leptin circulates at levels proportional to body fat; (ii) leptin entersthe CNS in proportion to its plasma concentration; (iii) leptinreceptors are found in brain neurons involved in regulating energyintake and expenditure; and (iv) leptin controls food intake and energyexpenditure by acting on receptors in the mediobasal hypothalmus.

It generally is believed that leptin inhibits the activity of neuronsthat contain neuropeptide Y (NPY) and agouti-related peptide (AgRP), andincreases the activity of neurons expressing α-melanocyte-stimulatinghormone (α-MSH). The NPY neurons are a key element in the regulation ofappetite; small doses of NPY injected into the brains of experimentalanimals stimulates feeding, while selective destruction of the NPYneurons in mice causes them to become anorexic. Conversely, α-MSH is animportant mediator of satiety, and differences in the gene for thereceptor at which α-MSH acts in the brain are linked to obesity inhumans.

It is not known how disturbances of production and aggregation of Aβpeptide give rise to the pathology of AD or other progressive cognitivedisorders. There remains a need for clinical therapy and diagnosticmethods of progressive cognitive disorders related to accumulation ofneurofibrillary tangles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows time- and dose-dependent dephosphorylation of tau by leptinin RA-SY5Y. Human neuroblastoma cells of the SY5Y cell line were inducedfor 7 days with retinoic acid (RA) (10 μM) to promote neuronaldifferentiation (RA-SY5Y). A. Induced cells were treated with leptin(400 ng/ml) for 4 hrs, or non-treated (placebo). Whole cell extractswere prepared and analyzed by western blot with anti-OB-R (leptinreceptor). Membranes were stripped and re-probed with anti-α-tubulin fornormalization. Representative blot is shown, n=3. B. Whole cell extractsfrom cells treated for various times with leptin (400 ng/ml), orplacebo, were prepared and analyzed by western blot with anti-tau(pSer³⁹⁶). Membranes were stripped and re-probed with anti-tau (total)for normalization. Representative blot is shown, n=3. C. Normalized banddensities from B were analyzed by densitometry. Results are presented asthe mean±SD percent fold change, relative to placebo-treated samples,which were arbitrarily assigned a value of 0. D. Induced cells weretreated with various concentrations of leptin for 4 hrs, or placebo.Experiments were then preformed as in B. E. Normalized band densititesfrom D were analyzed as in C. IC₅₀ represents the leptin concentrationat which tau (pSer³⁹⁶) phosphorylation is decreased by 50 percent.*p<0.05 vs. non-treated.

FIG. 2 shows time- and dose-dependent dephosphorylation of tau byinsulin in RA-SY5Y. A. RA-SY5Y were treated with insulin (10 μM) for 4hrs, or non-treated (placebo). Whole cell extracts were prepared andanalyzed by western blot with anti-insulin receptor (β-subunit).Membranes were stripped and re-probed with anti-α-tubulin fornormalization. Representative blot is shown, n=3. B. Whole cell extractsfrom cells treated for various times with insulin (10 μM), or placebo,were prepared and analyzed by western blot with anti-tau (pSer³⁹⁶).Membranes were stripped and re-probed with anti-tau (total) fornormalization. Representative blot is shown, n=3. C. Normalized banddensities from B were analyzed by densitometry. Results are presented asthe mean±SD percent fold change, relative to placebo-treated samples,which were arbitrarily assigned a value of 0. D. Induced cells weretreated with various concentrations of insulin for 4 hrs, or placebo.Experiments were then performed as in B. E. Normalized band densitiesfrom D were analyzed as in C. IC₅₀ represents the insulin concentrationat which tau (pSer³⁹⁶) phosphorylation is decreased by 50 percent.*p<0.05 vs. non-treated.

FIG. 3 shows that combined treatment of leptin and insulin produces agreater dephosphorylation of tau than either treatment alone. A. RA-SY5Ywere treated with low or high concentrations of leptin (100 or 1600ng/ml) and/or insulin (1 or 20 μM) for 4 hrs, or non-treated (placebo).Whole cell extracts were prepared and analyzed by western blot withanti-tau (pSer³⁹⁶). Membranes were stripped and re-probed with anti-tau(total) for normalization. Representative blot is shown, n=3. B.Normalized band densities from A were analyzed by densitometry. Resultsare presented as the mean±SD percent fold change, relative toplacebo-treated samples, which were arbitrarily assigned a value of 0.C. Cells were treated for 4 hrs with leptin (1600 ng/ml) and insulin (20μM), or placebo. To re-induce tau phosphorylation, cold PBS was added tothe post-treated cells for 10 min, 1 hr or not at all. Experiments werethen carried out as in A. D. Normalized band densitites from C wereanalyzed as in B. *p<0.05 vs. group. **p<0.01 vs. group.

FIG. 4 shows dephosphorylation of tau by 5′-AMP-activated protein kinase(AMPK) activation in RA-SY5Y. A. Induced cells were treated withaminoimidazole carboxamide ribonucleotide which acts as an AMP-activatedprotein kinase agonist (AICAR) (1 mM) for 1 hr, or non-treated(placebo). Whole cell extracts were prepared and analyzed by westernblot with AMPKα (pThr¹⁷²). Membranes were stripped and re-probed withanti-α-AMPKα (total) for normalization. Representative blot is shown,n=3. B. Whole cell extracts from cells treated for various times withAICAR (1 mM), or placebo, were prepared and analyzed by western blotwith anti-tau (pSer³⁹⁶). Membranes were stripped and re-probed withanti-tau (total) for normalization. Representative blot is shown, n=3.C. Normalized band densities from B were analyzed by densitometry.Results are presented as the mean±SD percent fold change, relative toplacebo-treated samples, which were arbitrarily assigned a value of 0.D. Induced cells were treated with various concentrations of AICAR for 1hr, or placebo. Experiments were then performed as in B. E. Normalizedband densitites from D were analyzed as in C. IC₅₀ represents the AICARconcentration at which tau (pSer³⁹⁶) phosphorylation is decreased by 50percent. *p<0.05 vs. non-treated.

SUMMARY

According to one aspect, the described invention provides a method fortreating a progressive cognitive disorder, the method comprising thestep of: (a) administering to a subject in need thereof a firstcomposition comprising (i) a phosphorylated tau accumulation-modulatingamount of a leptin composition, or a pharmaceutically acceptable saltthereof, and (ii) a pharmaceutically acceptable carrier, and (b)modulating accumulation of phosphorylated tau in cerebrospinal fluid ofthe subject. According to one embodiment of the method, the progressivecognitive disorder is selected from the group consisting of Alzheimer'sDisease, progressive supranuclear palsy, dementia, dementia pugilistica,Creutzfeldt-Jakob disease, frontotemporal dementia, Pick's disease, andFTDP-17 (parkinsonism) corticobasal degeneration. According to anotherembodiment, the leptin composition is a leptin, or a pharmaceuticallyacceptable salt thereof. According to another embodiment, the leptincomposition is a leptin mimic, or a pharmaceutically acceptable saltthereof. According to another embodiment, the leptin composition is aleptin derivative, or a pharmaceutically acceptable salt thereof.According to another embodiment, the leptin composition is a leptinagonist, or a pharmaceutically acceptable salt thereof. According toanother embodiment, the phosphorylated tau accumulation-modulatingamount is an amount from about 0.01 mg/kg body weight to about 100 mg/kgbody weight. According to another embodiment, the first compositionfurther comprises a second therapeutic agent. According to anotherembodiment, the second therapeutic agent is at least one of anantibiotic, an anti-fungal agent, an antiviral agent, an anti-protozoalagent, a steroidal anti-inflammatory agent, a non-steroidalanti-inflammatory agent, an anti-oxidant; a hormone; a vitamin; anantihistamine agent, and a chemotherapeutic agent. According to anotherembodiment, the progressive disorder comprises accumulation ofneurofibrillary tangles in brain.

According to another aspect, the described invention provides a methodfor improving resilience of cognitive function in a subject in needthereof, the method comprising the steps of (a) administering to thesubject a composition comprising (i) a cognitive function-enhancingamount of a leptin composition, and (ii) a pharmaceutically acceptablecarrier; and (b) modulating accumulation of phosphorylated tau incerebrospinal fluid of the subject. According to one embodiment of themethod, the leptin composition comprises at least one of a leptin, aleptin mimic, a leptin derivative, an AMP-dependent protein kinaseactivator, a leptin agonist, a leptin blocker, a mimic of a leptinblocker, a leptin antagonist, an AMP-dependent protein kinase inhibitor;or pharmaceutically acceptable salts thereof. According to anotherembodiment, the leptin composition further comprises a secondtherapeutic agent. According to another embodiment, the secondtherapeutic agent is at least one of an antibiotic, an anti-fungalagent, an antiviral agent, an anti-protozoal agent, a steroidalanti-inflammatory agent, a non-steroidal anti-inflammatory agent, ananti-oxidant; a hormone; a vitamin; an antihistamine agent, and achemotherapeutic agent.

DETAILED DESCRIPTION OF THE INVENTION

The described invention relates to methods for treating or preventing aprogressive cognitive disorder and methods for improving resilience ofcognitive function.

According to one aspect, the described invention provides a method fortreating a progressive cognitive disorder, the method comprising thesteps of: (a) administering to a subject in need thereof a firstcomposition comprising (i) a phosphorylated tau accumulation-modulatingamount of a leptin composition, and (ii) a pharmaceutically acceptablecarrier, and (b) modulating accumulation of phosphorylated tau. incerebrospinal fluid of the subject.

According to another aspect, the described invention provides a methodfor preventing progression of a progressive cognitive disorder, themethod comprising the steps of (a) administering to a subject in needthereof a first composition comprising (i) a phosphorylated tauaccumulation-modulating amount of a leptin composition, and (ii) apharmaceutically acceptable carrier, and (b) modulating accumulation ofphosphorylated tau in cerebrospinal fluid of the subject.

According to one embodiment, the leptin composition comprises a leptin,or a pharmaceutically acceptable salt thereof. According to anotherembodiment, the leptin composition comprises a leptin mimic, or apharmaceutically acceptable salt thereof. According to anotherembodiment, the leptin composition comprises a leptin derivative, or apharmaceutically acceptable salt thereof. According to anotherembodiment, the leptin composition comprises a leptin agonist, or apharmaceutically acceptable salt thereof. According to anotherembodiment, the leptin composition comprises an AMP-dependent proteinkinase activator, or a pharmaceutically acceptable salt thereof.According to another embodiment, the leptin composition comprises amimic of a leptin blocker, or a pharmaceutically acceptable saltthereof. According to another embodiment, the leptin compositioncomprises a leptin antagonist, or a pharmaceutically acceptable saltthereof. According to another embodiment, the leptin compositioncomprises an AMP-dependent protein kinase inhibitor, or apharmaceutically acceptable salt thereof.

According to another embodiment, the leptin composition comprises atleast one of a leptin, a leptin mimic, a leptin derivative, a leptinagonist, an AMP-dependent protein kinase activator, a mimic of a leptinblocker, a leptin antagonist, an AMP-dependent protein kinase inhibitor,or pharmaceutically acceptable salts thereof.

The term “treat” or “treating” as used herein refers to accomplishingone or more of the following: (a) reducing the severity of the disorder;(b) limiting development of symptoms characteristic of the disorder(s)being treated; (c) limiting worsening of symptoms characteristic of thedisorder(s) being treated; (d) limiting recurrence of the disorder(s) inpatients that have previously had the disorder(s); and (e) limitingrecurrence of symptoms in patients that were previously symptomatic forthe disorder(s).

The term “reduce” or “reducing” as used herein refers to limitoccurrence of the disorder in individuals at risk of developing thedisorder.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “disease” or “disorder” as used herein refers to an impairmentof health or a condition of abnormal functioning. The term “syndrome,”as used herein, refers to a pattern of symptoms indicative of somedisease or condition. The term “injury,” as used herein, refers todamage or harm to a structure or function of the body caused by anoutside agent or force, which may be physical or chemical. The term“condition”, as used herein, refers to a variety of health states and ismeant to include disorders or diseases caused by any underlyingmechanism or disorder, injury, and the promotion of healthy tissues andorgans.

Progressive cognitive disorders include, but are not limited to,progressive supranuclear palsy; dementia; dementia pugilistica; AD;Creutzfeldt-Jakob disease; frontotemporal dementia; Pick's disease;other tau-positive pathology including FTDP-17 (parkinsonism)corticobasal degeneration; frontotemporal lobar degeneration (FTLD);dementia lacking distinctive histology.

The term “administering” as used herein refers to causing to take orapportioning and includes in vivo administration, as well asadministration directly to tissue ex vivo. Generally, compositions maybe administered systemically either orally, buccally, parenterally,topically, by inhalation or insufflation (i.e., through the mouth orthrough the nose), or rectally in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired, or may be locally administered by means suchas, but not limited to, injection, implantation, grafting, topicalapplication, or parenterally.

The terms “subject” or “individual” or “patient” are usedinterchangeably to refer to a member of an animal species of mammalianorigin, including humans.

The phrase “a subject having a progressive cognitive disease” as usedherein refers to a subject who presents with diagnostic markers and/orsymptoms associated with a progressive cognitive disease. A progressivecognitive disease is usually diagnosed clinically from the patienthistory, collateral history from relatives, and clinical observations,based on the presence of characteristic neurological andneuropsychological features and the absence of alternative conditions.These criteria require that the presence of cognitive impairment, and asuspected dementia syndrome, be confirmed by neuropsychological testing.Advanced medical imaging with computed tomography (CT) or magneticresonance imaging (MRI), and with single photon emission computedtomography (SPECT) or positron emission tomography (PET) may be used tohelp exclude other cerebral pathology or subtypes of dementia.Assessment of intellectual functioning including memory testing canfurther characterize the state of the disease. A histopathologicconfirmation including a microscopic examination of brain tissue may berequired for a definitive diagnosis. For AD, eight cognitive domains aremost commonly impaired: memory, language, perceptual skills, attention,constructive abilities, orientation, problem solving and functionalabilities. These domains are equivalent to the NINCDS-ADRDA Alzheimer'sCriteria as listed in the Diagnostic and Statistical Manual of MentalDisorders (DSM-IV-TR) published by the American Psychiatric Association(incorporated in its entirety herein by reference).

A subject at risk of having a progressive cognitive disease is one whohas one or more predisposing factors to the development of a progressivecognitive disease.

A subject in need thereof is a patient having, or at risk of having, aprogressive cognitive disease.

The term “dementia” as used herein refers to a decline or a progressivedecline in cognitive function due to damage or disease in the brainbeyond what might be expected from normal aging. The term “cognitivefunction” refers to the intellectual processes resulting in anunderstanding, perception, or awareness of one's ideas as well as theability to perform mental tasks, such as thinking, learning, judging,remembering, computing, controlling motor functions, and the like.

The term “peptidomimetic” refers to a small protein-like chain designedto mimic or imitate a peptide. A peptidomimetic may comprisenon-peptidic structural elements capable of mimicking (meaningimitating) or antagonizing (meaning neutralizing or counteracting) thebiological action(s) of a natural parent peptide. The terms “leptinpeptidomimetic” “leptin mimic”, and “leptin mimetic” are usedinterchangeably herein to refer to a leptin derivative comprising afunctional domain of a leptin protein that produces a biological effect.In chemistry, a derivative is a compound that at least theoretically maybe formed from a precursor compound. These derivatives may be combinedwith another molecule to produce or enhance the biological effect. Thebiological effect may include, for example, but is not limited to,modulating amyloid peptide levels within a subject; modulating tauphosphorylation levels within a subject; decreasing amyloid peptidelevels within a subject; decreasing tau phosphorylation levels within asubject, and the like.

The term “antagonist” as used herein refers to a substance thatcounteracts the effects of another substance. The term “agonist” as usedherein refers to a chemical substance capable of activating a receptorto induce a full or partial pharmacological response. The term “blocker”as used herein refers to a substance that inhibits the physiologicalaction of another substance.

The term “leptin agonist” refers to a compound capable of activating theleptin receptor and/or downstream effectors and of modulating amyloidpeptide levels or tau phosphorylation in a subject. Such effectors mayinclude, for example, but are not limited to, AMP-dependent proteinkinase (“AMPK”) and sterol regulatory element binding proteins(“SREBP”).

The leptin receptor (OB-R), a member of the class I cytokine receptorsuperfamily, has at least six isoforms as a result of alternativesplicing. As used herein the term “isoform” refers to a version of aprotein that has the same function as another protein but that has somesmall difference(s) in its sequence. All isoforms of OB-R share anidentical extracellular ligand-binding domain. Leptin's functionalreceptor (OB-Rb), the b isoform, is expressed not only in thehypothalamus, where it regulates energy homeostasis and neuroendocrinefunction, but also in other brain regions and in the periphery,including all cell types of innate and adaptive immunity. Thefull-length b isoform (OB-Rb) lacks intrinsic tyrosine kinase activityand is involved in several downstream signal transduction pathways.

The terms “therapeutically effective amount”, an “amount effective”, or“pharmaceutically effective amount” of one or more of the active agentsare used interchangeably to refer to an amount that is sufficient toprovide the intended benefit of treatment. An effective amount of theactive agents that can be employed according to the described inventiongenerally ranges from generally about 0.01 mg/kg body weight to about100 g/kg body weight. However, dosage levels are based on a variety offactors, including the type of injury, the age, weight, sex, medicalcondition of the patient, the severity of the condition, the route ofadministration, and the particular active agent employed. Thus thedosage regimen may vary widely, but can be determined routinely by aphysician using standard methods. Additionally, the terms“therapeutically effective amounts” and “pharmaceutically effectiveamounts” include prophylactic or preventative amounts of thecompositions of the described invention. In prophylactic or preventativeapplications of the described invention, pharmaceutical compositions ormedicaments are administered to a patient susceptible to, or otherwiseat risk of, a disease, disorder or condition resulting from accumulationof an amyloid peptide in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the onset of the disease, disorderor condition, including biochemical, histologic and/or behavioralsymptoms of the disease, disorder or condition, its complications, andintermediate pathological phenotypes presenting during development ofthe disease, disorder or condition.

The term “phosphorylated tau accumulation modulating amount” as usedherein refers to a therapeutically effective amount of a leptincomposition that modulates the phosphorylation of tau protein. Aphosphorylated tau accumulation-modulating amount includes prophylacticor preventative amounts of the compositions of the described invention.

The term “cognitive function enhancing amount” as used herein refers toa therapeutically effective amount of a leptin composition (i.e., doseand frequency of administration) that modulates mental processes ofperception, memory, judgment or reasoning and thereby adds to, improves,or increases mental performance in a subject as compared to a subjectthat has not been administered a cognitive-function enhancing amount ofa composition or material.

A cognitive function enhancing amount is from about 0.01 mg/kg bodyweight to about 100 g/kg body weight.

According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 100 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 95 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 90 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 85 g/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 80 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 75 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 70 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 65 g/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 60 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 55 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 50 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 45 g/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 40 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 35 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 30 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 25 g/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 20 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 15 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 10 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 5 g/kg body weight. Accordingto another embodiment, the phosphorylated tau accumulation modulatingamount is from about 0.01 mg/kg body weight to about 4 g/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 3 g/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 2 g/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 1 g/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 500 mg/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 250mg/kg body weight. According to another embodiment, the phosphorylatedtau accumulation modulating amount is from about 0.01 mg/kg body weightto about 100 mg/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 50 mg/kg body weight. According to anotherembodiment, the phosphorylated tau accumulation modulating amount isfrom about 0.01 mg/kg body weight to about 25 mg/kg body weight.According to another embodiment, the phosphorylated tau accumulationmodulating amount is from about 0.01 mg/kg body weight to about 10 mg/kgbody weight. According to another embodiment, the phosphorylated tauaccumulation modulating amount is from about 0.01 mg/kg body weight toabout 5 mg/kg body weight. According to another embodiment, thephosphorylated tau accumulation modulating amount is from about 0.01mg/kg body weight to about 1 mg/kg body weight.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, composition or other substance that provides atherapeutic effect. The term “active” as used herein refers to theingredient, component or constituent of the compositions of the presentinvention responsible for the intended therapeutic effect. The terms“therapeutic agent” and “active agent” are used interchangeably herein.The active agent may be a therapeutically effective amount of at leastone of a leptin, a leptin mimic, a leptin derivative, or a leptinagonist or a pharmaceutically acceptable salt thereof.

The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e., dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED50, whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect may include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect also may include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

The term “drug” as used herein refers to a therapeutic agent or anysubstance, other than food, used in the prevention, diagnosis,alleviation, treatment, or cure of disease.

The compositions described herein are isolated molecules. An “isolatedmolecule” is a molecule that is substantially pure and is free of othersubstances with which it is ordinarily found in nature or in vivosystems to an extent practical and appropriate for its intended use. Inparticular, the compositions are sufficiently pure and are sufficientlyfree from other biological constituents of host cells so as to be usefulin, for example, producing pharmaceutical preparations or sequencing ifthe composition is a nucleic acid, peptide, or polysaccharide. Becausecompositions may be admixed with a pharmaceutically-acceptable carrierin a pharmaceutical preparation, the compositions may comprise only asmall percentage by weight of the preparation. The composition isnonetheless substantially pure in that it has been substantiallyseparated from the substances with which it may be associated in livingsystems or during synthesis. As used herein, the term “substantiallypure” refers purity of at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 99% pure as determined by ananalytical protocol. Such protocols may include, for example, but arenot limited to, FACS, HPLC, gel electrophoresis, chromatography, and thelike.

The leptin composition and/or the first composition may be combined withother therapeutic agents and administered locally. The leptincomposition and/or first composition and other therapeutic agent(s) maybe administered simultaneously or sequentially. When the othertherapeutic agents are administered simultaneously, they can beadministered in the same or separate formulations, but are administeredat the same time. The other therapeutic agents are administeredsequentially with one another and with leptin composition and/or firstcomposition when the administration of the other therapeutic agents andthe inhibitor is temporally separated. The separation in time betweenthe administration of these agents may be a matter of minutes or it maybe longer. The therapeutic agents may be a leptin antagonist, a leptinblocker, a leptin blocker, or an leptin antagonist, or combinationsthereof.

According to another embodiment, the leptin composition and/or the firstcomposition further comprises a second therapeutic agent. According tosome such embodiments, the second therapeutic agent is an antibioticagent. According to some such embodiments, the second therapeutic agentis an anti-fungal agent. According to some such embodiments, the secondtherapeutic agent is an anti-viral agent. According to some suchembodiments, the second therapeutic agent is an anti-protozoal agent.According to some such embodiments, the second therapeutic agent is asteroidal anti-inflammatory agent. According to some such embodiments,the second therapeutic agent is a non-steroidal anti-inflammatory agent.According to some such embodiments, the second therapeutic agent is ananti-oxidant agent. According to some such embodiments, the secondtherapeutic agent is a hormone. According to some such embodiments, thesecond therapeutic agent is a vitamin. According to some suchembodiments, the second therapeutic agent is an antihistamine agent.According to some such embodiments, the second therapeutic agent is achemotherapeutic agent.

The term “antibiotic agent” as used herein means any of a group ofchemical substances having the capacity to inhibit the growth of, or todestroy bacteria, and other microorganisms, used chiefly in thetreatment of infectious diseases. Examples of antibiotic agents include,but are not limited to, Penicillin G; Methicillin; Nafcillin; Oxacillin;Cloxacillin; Dicloxacillin; Ampicillin; Amoxicillin; Ticarcillin;Carbenicillin; Mezlocillin; Azlocillin; Piperacillin; Imipenem;Aztreonam; Cephalothin; Cefaclor; Cefoxitin; Cefuroxime; Cefonicid;Cefinetazole; Cefotetan; Cefprozil; Loracarbef; Cefetamet; Cefoperazone;Cefotaxime; Ceftizoxime; Ceftriaxone; Ceftazidime; Cefepime; Cefixime;Cefpodoxime; Cefsulodin; Fleroxacin; Nalidixic acid; Norfloxacin;Ciprofloxacin; Ofloxacin; Enoxacin; Lomefloxacin; Cinoxacin;Doxycycline; Minocycline; Tetracycline; Amikacin; Gentamicin; Kanamycin;Netilmicin; Tobramycin; Streptomycin; Azithromycin; Clarithromycin;Erythromycin; Erythromycin estolate; Erythromycin ethyl succinate;Erythromycin glucoheptonate; Erythromycin lactobionate; Erythromycinstearate; Vancomycin; Teicoplanin; Chloramphenicol; Clindamycin;Trimethoprim; Sulfamethoxazole; Nitrofurantoin; Rifampin; Mupirocin;Metronidazole; Cephalexin; Roxithromycin; Co-amoxiclavuanate;combinations of Piperacillin and Tazobactam; and their various salts,acids, bases, and other derivatives. Anti-bacterial antibiotic agentsinclude, but are not limited to, penicillins, cephalosporins,carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides,glycopeptides, quinolones, tetracyclines, macrolides, andfluoroquinolones.

The term “anti-fungal agent” as used herein means any of a group ofchemical substances having the capacity to inhibit the growth of or todestroy fungi. Anti-fungal agents include but are not limited toAmphotericin B, Candicidin, Dermostatin, Filipin, Fungichromin,Hachimycin, Hamycin, Lucensomycin, Mepartricin, Natamycin, Nystatin,Pecilocin, Perimycin, Azaserine, Griseofulvin, Oligomycins, Neomycin,PyrroInitrin, Siccanin, Tubercidin, Viridin, Butenafine, Naftifine,Terbinafine, Bifonazole, Butoconazole, Chlordantoin, Chlormidazole,Cloconazole, Clotrimazole, Econazole, Enilconazole, Fenticonazole,Flutrimazole, Isoconazole, Ketoconazole, Lanoconazole, Miconazole,Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole,Tolciclate, Tolindate, Tolnaftate, Fluconawle, Itraconazole,Saperconazole, Terconazole, Acrisorcin, Amorolfine, Biphenamine,Bromosalicylchloranilide, Buclosamide, Calcium Propionate,Chlorphenesin, Ciclopirox, Cloxyquin, Coparaffinate, Diamthazole,Exalamide, Flucytosine, Halethazole, Hexetidine, Loflucarban, Nifuratel,Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, SodiumPropionate, Sulbentine, Tenonitrozole, Triacetin, Ujothion, UndecylenicAcid, and Zinc Propionate.

The term “anti-viral agent” as used herein means any of a group ofchemical substances having the capacity to inhibit the replication of orto destroy viruses used chiefly in the treatment of viral diseases.Anti-viral agents include, but are not limited to, Acyclovir, Cidofovir,Cytarabine, Dideoxyadenosine, Didanosine, Edoxudine, Famciclovir,Floxuridine, Ganciclovir, Idoxuridine, Inosine Pranobex, Lamivudine,MADU, Penciclovir, Sorivudine, Stavudine, Trifluridine, Valacyclovir,Vidarabine, Zalcitabine, Zidovudine, Acemannan, Acetylleucine,Amantadine, Amidinomycin, Delavirdine, Foscamet, Indinavir, Interferons(e.g., IFN-alpha), Kethoxal, Lysozyme, Methisazone, Moroxydine,Nevirapine, Podophyllotoxin, Ribavirin, Rimantadine, Ritonavir2,Saquinavir, Stailimycin, Statolon, Tromantadine, Zidovudine (AZT) andXenazoic Acid.

The term “anti-protozoal agent” as used herein means any of a group ofchemical substances having the capacity to inhibit the growth of or todestroy protozoans used chiefly in the treatment of protozoal diseases.Examples of antiprotozoal agents, without limitation includepyrimethamine (Daraprim®) sulfadiazine, and Leucovorin.

“Steroidal anti-inflammatory agent”, as used herein, refer to any one ofnumerous compounds containing a 17-carbon 4-ring system and includes thesterols, various hormones (as anabolic steroids), and glycosides.Representative examples of steroidal anti-inflammatory drugs include,without limitation, corticosteroids such as hydrocortisone,hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, chlorprednisone acetate,clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide,flunisolide, fluoromethalone, fluperolone, fluprednisolone,hydrocortisone valerate, hydrocortisone cyclopentylpropionate,hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone,beclomethasone dipropionate, triamcinolone, and mixtures thereof.

“Non-steroidal anti-inflammatory agents” refers to a large group ofagents that are aspirin-like in their action, including ibuprofen(Advil)®, naproxen sodium (Aleve)®, and acetaminophen (Tylenol)®.Additional examples of non-steroidal anti-inflammatory agents that areusable in the context of the present invention include, withoutlimitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam,and CP-14,304; disalcid, benorylate, trilisate, safapryn, solprin,diflunisal, and fendosal; acetic acid derivatives, such as diclofenac,fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac,tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac,oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic,meclofenamic, flufenamic, niflumic, and tolfenamic acids; propionic acidderivatives, such as ibuprofen, naproxen, benoxaprofen, flurbiprofen,ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone,feprazone, azapropazone, and trimethazone. Mixtures of thesenon-steroidal anti-inflammatory agents may also be employed, as well asthe dermatologically acceptable salts and esters of these agents. Forexample, etofenamate, a flufenamic acid derivative, is particularlyuseful for topical application.

“An anti-oxidant agent” as used herein refers to a substance thatinhibits oxidation or reactions promoted by oxygen or peroxides.Non-limiting examples of anti-oxidants that are usable in the context ofthe present invention include ascorbic acid (vitamin C) and its salts,ascorbyl esters of fatty acids, ascorbic acid derivatives (e.g.,magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbylsorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherolacetate, other esters of tocopherol, butylated hydroxy benzoic acids andtheir salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(commercially available under the tradename Trolox®), gallic acid andits alkyl esters, especially propyl gallate, uric acid and its salts andalkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g.,N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g.,glutathione), dihydroxy fumaric acid and its salts, glycine pidolate,arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin,lysine, methionine, proline, superoxide dismutase, silymarin, teaextracts, grape skin/seed extracts, melanin, and rosemary extracts.

“Chemotherapeutic agent” refers to chemicals useful in the treatment orcontrol of a disease. Non-limiting examples of chemotherapeutic agentsusable in context of the present invention include daunorubicin,doxorubicin, idarubicin, amrubicin, pirarubicin, epirubicin,mitoxantrone, etoposide, teniposide, vinblastine, vincristine, mitomycinC, 5-FU, paclitaxel, docetaxel, actinomycin D, colchicine, topotecan,irinotecan, gemcitabine cyclosporin, verapamil, valspodor, probenecid,MK571, GF120918, LY335979, biricodar, terfenadine, quinidine,pervilleine A and XR9576.

“Antihistamine agent” as used herein refers to any of various compoundsthat counteract histamine in the body and that are used for treatingallergic reactions (such as hay fever) and cold symptoms. Non-limitingexamples of antihistamines usable in context of the present inventioninclude chlorpheniramine, brompheniramine, dexchlorpheniramine,tripolidine, clemastine, diphenhydramine, promethazine, piperazines,piperidines, astemizole, loratadine and terfenadine.

“Vitamin” as used herein, refers to any of various organic substancesessential in minute quantities to the nutrition of most animals actespecially as coenzymes and precursors of coenzymes in the regulation ofmetabolic processes. Non-limiting examples of vitamins usable in contextof the present invention include vitamin A and its analogs andderivatives: retinol, retinal, retinol palmitate, retinoic acid,tretinoin, iso-tretinoin (known collectively as retinoids), vitamin E(tocopherol and its derivatives), vitamin C (L-ascorbic acid and itsesters and other derivatives), vitamin B₃ (niacinamide and itsderivatives), alpha hydroxy acids (such as glycolic acid, lactic acid,tartaric acid, malic acid, citric acid, etc.) and beta hydroxy acids(such as salicylic acid and the like).

“Hormone” as used herein refers to natural substances produced by organsof the body that travel by blood to trigger activity in other locationsor their synthetic analogs. Suitable hormones for use in the context ofthe present invention include, but are not limited to, any hormoneproduced by neurosecretory cells, including gonadotropin releasinghormone (GnRH), corticotropin releasing hormone (CRH), thyrotropinreleasing hormone (TRH), prolactin inhibiting hormone (dopamine) andorexin (hypocretin), as well as recombinant hormones, meaning hormonesproduced by a process using DNA engineered to contain sequences thatnormally would not occur together and introducing that DNA into thecells of a host.

Neurofibrillary tangles (“NFT”) generally refer to aggregates of themicrotubule-associated protein “tau”, which have becomehyperphosphorylated and accumulate inside the cells themselves.

According to one embodiment, the progressive cognitive disordercomprises accumulation of neurofibrillary tangles in brain. According toanother embodiment, the progressive cognitive disorder is Alzheimer'sDisease. According to another embodiment, progressive cognitive disorderis progressive supranuclear palsy. According to another embodiment,progressive cognitive disorder is dementia. According to anotherembodiment, progressive cognitive disorder is dementia pugilistica.According to another embodiment, progressive cognitive disorder isCreutzfeldt-Jakob disease. According to another embodiment, progressivecognitive disorder is frontotemporal dementia. According to anotherembodiment, progressive cognitive disorder is Pick's disease. Accordingto another embodiment, progressive cognitive disorder is FTDP-17(parkinsonism) corticobasal degeneration. According to another aspect,the present invention provides a method of improving resilience ofcognitive function in a subject in need thereof, the method comprisingthe step of (a) administering to the subject a composition comprising:(i) a cognitive function-enhancing amount of a leptin composition; and(ii) a pharmaceutically acceptable carrier; and (b) modulatingaccumulation of phosphorylated tau in cerebrospinal fluid of thesubject.

The term “resilience” as used herein refers to the ability to return tothe original form, position, or function after or during an illness,condition, disease, syndrome or disorder.

According to one embodiment of the method, the leptin compositioncomprises a leptin, or a pharmaceutically acceptable salt thereof.According to another embodiment, the leptin composition comprises aleptin mimic, or a pharmaceutically acceptable salt thereof. Accordingto another embodiment, the leptin composition comprises a leptinderivative, or a pharmaceutically acceptable salt thereof. According toanother embodiment, the leptin composition comprises an AMP-dependentprotein kinase activator, or a pharmaceutically acceptable salt thereof.According to another embodiment, the leptin composition comprises aleptin agonist, or a pharmaceutically acceptable salt thereof. Accordingto another embodiment, the leptin composition comprises a leptinblocker, or a pharmaceutically acceptable salt thereof. According toanother embodiment, the leptin composition comprises a mimic of a leptinblocker, or a pharmaceutically acceptable salt thereof. According toanother embodiment, the leptin composition comprises a leptinantagonist, or a pharmaceutically acceptable salt thereof. According toanother embodiment, the leptin composition comprises an AMP-dependentprotein kinase inhibitor.

According to another embodiment, the leptin composition furthercomprises a second therapeutic agent. According to some suchembodiments, the second therapeutic agent is an antibiotic. According tosome such embodiments, the second therapeutic agent is an anti-fungalagent. According to some such embodiments, the second therapeutic agentis an anti-viral agent. According to some such embodiments, the secondtherapeutic agent is an anti-protozoal agent. According to some suchembodiments, the second therapeutic agent is a non-steroidalanti-inflammatory agent. According to some such embodiments, the secondtherapeutic agent is an anti-oxidant. According to some suchembodiments, the second therapeutic agent is a steroidalanti-inflammatory agent. According to some such embodiments, the secondtherapeutic agent is a hormone. According to some such embodiments, thesecond therapeutic agent is a vitamin. According to some suchembodiments, the second therapeutic agent is an antihistamine agent.According to some such embodiments, the second therapeutic agent is anchemotherapeutic agent.

Compositions

The compositions are delivered in therapeutically effective amounts.Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective prophylactic ortherapeutic treatment regimen may be planned which does not causesubstantial toxicity and yet is effective to treat the particularsubject. The effective amount for any particular application may varydepending on such factors as the disease or condition being treated, theparticular therapeutically active leptin, leptin mimic, leptin agonist,leptin derivative peptide, leptin blocker and/or leptin antagonist, orcombinations thereof, being administered, the size of the subject, orthe severity of the disease or condition. One of ordinary skill in theart may determine empirically the effective amount of a particularleptin composition and/or other therapeutic agent without necessitatingundue experimentation. It generally is preferred that a maximum dose beused, that is, the highest safe dose according to some medical judgment.“Dose” and “dosage” are used interchangeably herein.

For any compound described herein the therapeutically effective amountinitially may be determined from preliminary in vitro studies and/oranimal models. A therapeutically effective dose may also be determinedfrom human data for a therapeutically active leptin, a leptin mimic, aleptin agonist, a leptin derivative peptide, a leptin blocker and/or aleptin antagonist, or combinations thereof, which have been tested inhumans and for compounds which are known to exhibit similarpharmacological activities, such as other related active agents. Theapplied dose may be adjusted based on the relative bioavailability andpotency of the administered compound or composition. Adjusting the doseto achieve maximal efficacy based on the methods described above andother methods as are well-known in the art is well within thecapabilities of the ordinarily skilled artisan.

The formulations of a first composition, a leptin composition, atherapeutically active leptin, a leptin mimic, a leptin agonist, aleptin derivative peptide, a leptin blocker and/or a leptin antagonist,or combinations thereof, may be administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the first composition, and/ora leptin composition, a therapeutically active leptin, a leptin mimic, aleptin agonist, a leptin derivative peptide, a leptin blocker and/or aleptin antagonist, or combinations thereof, may be administered to asubject by any mode that delivers the leptin composition and/or thefirst composition to the desired surface. Administering thepharmaceutical composition may be accomplished by any means known to theskilled artisan. Routes of administration include, but are not limitedto, intrathecal, intra-arterial, parenteral (e.g. intravenous), orintramuscular, orally, buccally, intranasally, rectally, or topically.

The inhibitors and other therapeutics may be delivered to a subjectduring surgery to treat an underlying condition or side effect such assubarachnoid hemorrhage or peripheral vasospasm or during intra-arterialprocedures.

Oral Compositions

The compositions of the present invention may be in a form suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules or syrups or elixirs. As used herein, the terms “oral” or“orally” refer to the introduction into the body by mouth wherebyabsorption occurs in one or more of the following areas of the body: themouth, stomach, small intestine, lungs (also specifically referred to asinhalation), and the small blood vessels under the tongue (alsospecifically referred to as sublingually). Compositions intended fororal use may be prepared according to any known method, and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents, andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets may contain the active ingredient(s) inadmixture with non-toxic pharmaceutically-acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be,for example, inert diluents, such as calcium carbonate, sodiumcarbonate, lactose, calcium phosphate or sodium phosphate; granulatingand disintegrating agents, for example, corn starch or alginic acid;binding agents, for example, starch, gelatin or acacia; and lubricatingagents, for example, magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They also may be coated for controlled release.

Compositions of the present invention also may be formulated for oraluse as hard gelatin capsules, where the active ingredient(s) is(are)mixed with an inert solid diluent, for example, calcium carbonate,calcium phosphate or kaolin, or soft gelatin capsules wherein the activeingredient(s) is (are) mixed with water or an oil medium, for example,peanut oil, liquid paraffin, or olive oil.

The compositions of the present invention may be formulated as aqueoussuspensions wherein the active ingredient(s) is (are) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatidesuch as lecithin, or condensation products of an alkylene oxide withfatty acids, for example, polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample, heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions also may contain one or more coloring agents,one or more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Compositions of the present invention may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil, forexample arachis oil, olive oil, sesame oil or coconut oil, or in amineral oil, such as liquid paraffin. The oily suspensions may contain athickening agent, for example, beeswax, hard paraffin or cetyl alcohol.Sweetening agents, such as those set forth above, and flavoring agentsmay be added to provide a palatable oral preparation. These compositionsmay be preserved by the addition of an antioxidant such as ascorbicacid.

Compositions of the present invention may be formulated in the form ofdispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water. The active ingredient in suchpowders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients, for example,sweetening, flavoring and coloring agents, also may be present.

The compositions of the invention also may be in the form of anemulsion. An emulsion is a two-phase system prepared by combining twoimmiscible liquid carriers, one of which is disbursed uniformlythroughout the other and consists of globules that have diameters equalto or greater than those of the largest colloidal particles. The globulesize is critical and must be such that the system achieves maximumstability. Usually, separation of the two phases will not occur unless athird substance, an emulsifying agent, is incorporated. Thus, a basicemulsion contains at least three components, the two immiscible liquidcarriers and the emulsifying agent, as well as the active ingredient.Most emulsions incorporate an aqueous phase into a non-aqueous phase (orvice versa). However, it is possible to prepare emulsions that arebasically non-aqueous, for example, anionic and cationic surfactants ofthe non-aqueous immiscible system glycerin and olive oil. Thus, thecompositions of the invention may be in the form of an oil-in-wateremulsion. The oily phase may be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example a liquid paraffin, or amixture thereof. Suitable emulsifying agents may be naturally-occurringgums, for example, gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of the partial esterswith ethylene oxide, for example, polyoxyethylene sorbitan monooleate.The emulsions also may contain sweetening and flavoring agents.

The compositions of the invention also may be formulated as syrups andelixirs. Syrups and elixirs may be formulated with sweetening agents,for example, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations also may contain a demulcent, a preservative, and flavoringand coloring agents. Demulcents are protective agents employed primarilyto alleviate irritation, particularly mucous membranes or abraded(meaning torn or cut) tissues. A number of chemical substances possessdemulcent properties. These substances include the alginates, mucilages,gums, dextrins, starches, certain sugars, and polymeric polyhydricglycols. Others include acacia, agar, benzoin, carbomer, gelatin,glycerin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, propylene glycol, sodium alginate, tragacanth,hydrogels and the like.

Buccal Compositions

For buccal administration, the compositions of the present invention maytake the form of tablets or lozenges formulated in a conventionalmanner.

Parenteral Compositions

The compositions of the present invention may be in the form of asterile injectable aqueous or oleaginous suspension. The term“parenteral” as used herein refers to introduction into the body by wayof an injection (i.e., administration by injection), including, forexample, subcutaneously (i.e., an injection beneath the skin),intramuscularly (i.e., an injection into a muscle); intravenously (i.e.,an injection into a vein), intrathecally (i.e., an injection into thespace around the spinal cord), intrastemal injection, or infusiontechniques. A parenterally administered composition of the presentinvention is delivered using a needle, e.g., a surgical needle. The term“surgical needle” as used herein, refers to any needle adapted fordelivery of fluid (i.e., capable of flow) compositions of the presentinvention into a selected anatomical structure. Injectable preparations,such as sterile injectable aqueous or oleaginous suspensions, may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents.

The first composition and/or leptin composition, therapeutically activeleptin, leptin mimic, leptin agonist, leptin derivative peptide, leptinblocker and/or leptin antagonist, when it is desirable to deliver themlocally, may be formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, for example, in ampoulesor in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilssuch as sesame oil, or synthetic fatty acid esters, such as ethyl oleateor triglycerides, or liposomes. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension also may contain suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Alternatively, the active compounds maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, microencapsulated, and if appropriate, with one or moreexcipients, encochleated, coated onto microscopic gold particles,contained in liposomes, pellets for implantation into the tissue, ordried onto an object to be rubbed into the tissue. Such pharmaceuticalcompositions also may be in the form of granules, beads, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, drops or preparations with protractedrelease of active compounds, in whose preparation excipients andadditives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, or solubilizers are customarilyused as described above. The pharmaceutical compositions are suitablefor use in a variety of drug delivery systems. For a brief review ofmethods for drug delivery, see Langer 1990 Science 249, 1527-1533, whichis incorporated herein by reference.

The first composition and/or leptin composition, therapeutically activeleptin, leptin mimic, leptin agonist, leptin derivative peptide, leptinblocker and/or leptin antagonist, or combinations thereof, andoptionally other therapeutics may be administered per se (neat) or inthe form of a pharmaceutically acceptable salt. Other therapeutics mayinclude, but are not limited to, an antibiotic agent, an anti-fungalagent, an anti-viral agent, an anti-protozoal agent, a steroidalanti-inflammatory agent, a non-steroidal anti-inflammatory agent, ananti-oxidant agent, a hormone, a vitamin, an antihistamine agent, achemotherapetic agent, or combinations thereof. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts may be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group. By “pharmaceutically acceptable salt” is meantthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well-known in the art. For example, P. H. Stahl, etal. describe pharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley V C H,Zurich, Switzerland: 2002). The salts may be prepared in situ during thefinal isolation and purification of the compounds described within thepresent invention or separately by reacting a free base function with asuitable organic acid. Representative acid addition salts include, butare not limited to, acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsalso may be obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsalso may be made.

The formulations may be presented conveniently in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association a leptincomposition, a therapeutically active leptin, a leptin mimic, a leptinagonist, a leptin derivative peptide, a leptin blocker and/or a leptinantagonist, or combinations thereof, or a pharmaceutically acceptablesalt or solvate thereof (“active compound”) with the carrier whichconstitutes one or more accessory agents. In general, the formulationsare prepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation.

The pharmaceutical agent or a pharmaceutically acceptable ester, salt,solvate or prodrug thereof may be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action. Solutions or suspensions used for parenteral,intradermal, subcutaneous, intrathecal, or topical application mayinclude, but are not limited to, for example, the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parental preparationmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Administered intravenously, particularcarriers are physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity may be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants including preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release may be controlled.Such long acting formulations may be formulated with suitable polymericor hydrophobic materials (for example as an emulsion in an acceptableoil) or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissues.

The locally injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions may be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral (including but not limited to, subcutaneous,intradermal, intramuscular, intravenous, intrathecal and intraarticular)administration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes, which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline, water-for-injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Another method of formulation of the compositions described hereininvolves conjugating the compounds described herein to a polymer thatenhances aqueous solubility. Examples of suitable polymers include butare not limited to polyethylene glycol, poly-(d-glutamic acid),poly-(1-glutamic acid), poly-(1-glutamic acid), poly-(d-aspartic acid),poly-(1-aspartic acid), poly-(1-aspartic acid) and copolymers thereof.Polyglutamic acids having molecular weights between about 5,000 to about100,000, with molecular weights between about 20,000 and about 80,000may be used and with molecular weights between about 30,000 and about60,000 may also be used.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The therapeutic agent(s), including the leptin composition,therapeutically active leptin, leptin mimic, leptin agonist, leptinderivative peptide, leptin blocker and/or leptin antagonist, orcombinations thereof, may be provided in particles. The term “particles”as used herein refers to nano- or microparticles (or in some instanceslarger) that may contain in whole or in part the leptin composition,therapeutically active leptin, leptin mimic, leptin agonist, leptinderivative peptide, leptin blocker and/or leptin antagonist, orcombinations thereof, or the other therapeutic agent(s) as describedherein, including, but not limited to, an antibiotic agent, ananti-fungal agent, an anti-viral agent, an anti-protozoal agent, asteroidal anti-inflammatory agent, a non-steroidal anti-inflammatoryagent, an anti-oxidant agent, a hormone, a vitamin, an antihistamineagent, a chemotherapetic agent, or combinations thereof. The particlesmay contain the therapeutic agent(s) in a core surrounded by a coating.The therapeutic agent(s) also may be dispersed throughout the particles.The therapeutic agent(s) also may be adsorbed into the particles. Theparticles may be of any order release kinetics, including zero orderrelease, first order release, second order release, delayed release,sustained release, immediate release, etc., and any combination thereof.The particle may include, in addition to the therapeutic agent(s), anyof those materials routinely used in the art of pharmacy and medicine,including, but not limited to, erodible, nonerodible, biodegradable, ornonbiodegradable material or combinations thereof. The particles may bemicrocapsules that contain the leptin composition, therapeuticallyactive leptin, leptin mimic, leptin agonist, leptin derivative peptide,leptin blocker and/or leptin antagonist, or combinations thereof, in asolution or in a semi-solid state. The particles may be of virtually anyshape.

Both non-biodegradable and biodegradable polymeric materials may be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired.Bioadhesive polymers of particular interest include bioerodiblehydrogels as described by Sawhney et al in Macromolecules (1993) 26,581-587, the teachings of which are incorporated herein. These includepolyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), andpoly(octadecyl acrylate).

Insufflation Compositions

The compositions of the present invention may be in the form of adispersible dry powder for delivery by inhalation or insufflation(either through the mouth or through the nose). Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures ofwhich are incorporated by reference. Spray drying, for example, is aprocess in which a homogeneous aqueous mixture of drug and the carrieris introduced via a nozzle (e.g., a two fluid nozzle), spinning disc oran equivalent device into a hot gas stream to atomize the solution toform fine droplets. The aqueous mixture may be a solution, suspension,slurry, or the like, but needs to be homogeneous to ensure uniformdistribution of the components in the mixture and ultimately thepowdered composition. The solvent, generally water, rapidly evaporatesfrom the droplets producing a fine dry powder having particles fromabout 1 μm to 5 μm in diameter. The spray drying is done underconditions that result in a substantially amorphous powder ofhomogeneous constitution having a particle size that is respirable, alow moisture content and flow characteristics that allow for readyaerosolization. Preferably the particle size of the resulting powder issuch that more than about 98% of the mass is in particles having adiameter of about 10 μm or less with about 90% of the mass being inparticles having a diameter less than 5 μm. Alternatively, about 95% ofthe mass will have particles with a diameter of less than 10 μm withabout 80% of the mass of the particles having a diameter of less than 5μm. Dry powder compositions also may be prepared by lyophilization andjet milling, as disclosed in International Patent Publication No. WO91/16038, the disclosure of which are incorporated by reference.

The term “dispersibility” or “dispersible” means a dry powder having amoisture content of less than about 10% by weight (% w) water, usuallybelow about 5% w and preferably less than about 3% w; a particle size ofabout 1.0-5.0 μm mass median diameter (MMD), usually 1.0-4.0 μm MMD, andpreferably 1.0-3.0 μm MMD; a delivered dose of about >30%, usually >40%,preferably >50%, and most preferred>60%; and an aerosol particle sizedistribution of about 1.0-5.0 μm mass median aerodynamic diameter(MMAD), usually 1.5-4.5 μm MMAD, and preferably 1.5-4.0 μm MMAD. Methodsand compositions for improving dispersibility are disclosed in U.S.application Ser. No. 08/423,568, filed Apr. 14, 1995, the disclosure ofwhich is hereby incorporated by reference.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 microns (μm) in diameterwith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content suchthat the particles are readily dispersible in an inhalation device toform an aerosol. This moisture content is generally below about 10% byweight (% w) water, usually below about 5% w and preferably less thanabout 3% w.

The amount of the pharmaceutically acceptable carrier is that amountneeded to provide the necessary stability, dispersibility, consistencyand bulking characteristics to ensure a uniform pulmonary delivery ofthe composition to a subject in need thereof. Numerically the amount maybe from about 0.05% w to about 99.95% w, depending on the activity ofthe drug being employed. Preferably about 5% w to about 95% will beused. The carrier may be one or a combination of two or morepharmaceutical excipients, but generally will be substantially free ofany “penetration enhancers.” Penetration enhancers are surface activecompounds which promote penetration of a drug through a mucosal membraneor lining and are proposed for use in intranasal, intrarectal, andintravaginal drug formulations. Exemplary penetration enhancers includebile salts, e.g., taurocholate, glycocholate, and deoxycholate;fusidates, e.g., taurodehydrofusidate; and biocompatible detergents,e.g., Tweens, Laureth-9, and the like. The use of penetration enhancersin formulations for the lungs, however, is generally undesirable becausethe epithelial blood barrier in the lung can be adversely affected bysuch surface active compounds. The dry powder compositions of thepresent invention are readily absorbed in the lungs without the need toemploy penetration enhancers.

The types of pharmaceutical excipients that are useful as carriers forpulmonary delivery include stabilizers such as human serum albumin(HSA), bulking agents such as carbohydrates, amino acids andpolypeptides; pH adjusters or buffers; salts such as sodium chloride;and the like. These carriers may be in a crystalline or amorphous formor may be a mixture of the two.

Bulking agents that are particularly valuable for pulmonary deliveryinclude compatible carbohydrates, polypeptides, amino acids orcombinations thereof. Suitable carbohydrates include monosaccharidessuch as galactose, D-mannose, sorbose, and the like; disaccharides, suchas lactose, trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such as raffinose,maltodextrins, dextrans, and the like; alditols, such as mannitol,xylitol, and the like. A preferred group of carbohydrates includeslactose, trehalose, raffinose, maltodextrins, and mannitol. Suitablepolypeptides include aspartame. Amino acids include alanine and glycine,with glycine being preferred.

Additives, which are minor components of the composition for pulmonarydelivery, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

For delivery by inhalation or insufflation, the composition of thepresent invention is placed within a suitable dosage receptacle in anamount sufficient to provide a subject with a unit dosage treatment. Thedosage receptacle is one that fits within a suitable inhalation deviceto allow for the aerosolization of the dry powder composition bydispersion into a gas stream to form an aerosol and then capturing theaerosol so produced in a chamber having a mouthpiece attached forsubsequent inhalation by a subject in need of treatment. Such a dosagereceptacle includes any container enclosing the composition known in theart such as gelatin or plastic capsules with a removable portion thatallows a stream of gas (for example, air) to be directed into thecontainer to disperse the dry powder composition. Such containers areexemplified by those shown in U.S. Pat. No. 4,227,522; U.S. Pat. No.4,192,309; and U.S. Pat. No. 4,105,027. Suitable containers also includethose used in conjunction with Glaxo's Ventolin® Rotohaler brand powderinhaler or Fison's Spinhaler® brand powder inhaler. Another suitableunit-dose container which provides a superior moisture barrier is formedfrom an aluminum foil plastic laminate. The pharmaceutical-based powderis filled by weight or by volume into the depression in the formablefoil and hermetically sealed with a covering foil-plastic laminate. Sucha container for use with a powder inhalation device is described in U.S.Pat. No. 4,778,054 and is used with Glaxo's Diskhaler (U.S. Pat. Nos.4,627,432; 4,811,731; and 5,035,237). All of these references areincorporated herein by reference.

The compositions of the invention may be used in the form of drops orsprays (e.g., a nasal spray, aerosol spray, or pump spray) or othervehicles for nasal administration (intranasal delivery). Aerosol spraypreparations can be contained in a pressurized container with a suitablepropellant such as a hydrocarbon propellant. Pump spray dispensers candispense a metered dose or a dose having a specific particle or dropletsize. Any dispensing device can be arranged to dispense only a singledose, or a multiplicity of doses. More generally, compositions of theinvention, especially those formulated for intranasal administration,can also be provided as solutions, suspensions, or viscous compositions(e.g., gels, lotions, creams, or ointments).

Rectal Compositions

The compositions of the present invention may be in the form ofsuppositories for rectal administration of the composition. “Rectal” or“rectally” as used herein refers to introduction into the body throughthe rectum where absorption occurs through the walls of the rectum.These compositions can be prepared by mixing the drug with a suitablenonirritating excipient such as cocoa butter and polyethylene glycolswhich are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.When formulated as a suppository the compositions of the invention maybe formulated with traditional binders and carriers, such astriglycerides.

Topical Compositions

The term “topical” refers to administration of an inventive compositionat, or immediately beneath, the point of application. The phrase“topically applying” describes application onto one or more surfaces(s)including epithelial surfaces. Although topical administration, incontrast to transdermal administration, generally provides a localrather than a systemic effect, as used herein, unless otherwise statedor implied, the terms topical administration and transdermaladministration are used interchangeably. For the purpose of thisapplication, topical applications shall include mouthwashes and gargles.

Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis deviceswhich are prepared according to techniques and procedures well known inthe art. The terms “transdermal delivery system,” “transdermal patch” or“patch” refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for post-menopausalindications, and nicotine for smoking cessation.

Patches suitable for use in the present invention include, but are notlimited to, (1) the matrix patch; (2) the reservoir patch; (3) themulti-laminate drug-in-adhesive patch; and (4) the monolithicdrug-in-adhesive patch; Transdermal and Topical Drug Delivery Systems,pp. 249-297 (Tapash K. Ghosh et al. eds., 1997), hereby incorporatedherein by reference. These patches are well known in the art andgenerally available commercially.

Carriers and Other Components

In some embodiments, the compositions of the present invention may beformulated with an excipient, vehicle or carrier selected from solvents,suspending agents, binding agents, fillers, lubricants, disintegrants,and wetting agents/surfactants/solubilizing agents. The terms“excipient”, “vehicle”, or “carrier” refer to substances that facilitatethe use of, but do not deleteriously react with, the active compound(s)when mixed with it. The term “active” refers to the ingredient,component or constituent of the compositions of the present inventionresponsible for the intended therapeutic effect. Carriers must be ofsufficiently high purity and of sufficiently low toxicity to render themsuitable for administration to the subject being treated. The carriercan be inert, or it can possess pharmaceutical benefits.

The carrier can be liquid or solid and is selected with the plannedmanner of administration in mind to provide for the desired bulk,consistency, etc., when combined with an active and the other componentsof a given composition. Typical pharmaceutical carriers include, but arenot limited to, binding agents (including, but not limited to,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (including, but not limited to, lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate.);lubricants (including, but not limited to, magnesium stearate, talc,silica, sollidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate); disintegrants (including, but not limited to,starch, sodium starch glycolate) and wetting agents (including, but notlimited to, sodium lauryl sulfate). Additional suitable carriers for thecompositions of the present invention include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil; fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, and the like. The pharmaceutical preparations canbe sterilized and if desired, mixed with auxiliary agents, for example,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds.

The term “pharmaceutically acceptable carrier” as used herein refers toany substantially non-toxic carrier conventionally useful foradministration of pharmaceuticals in which the active component willremain stable and bioavailable. In some embodiments, thepharmaceutically acceptable carrier of the compositions of the presentinvention include a release agent such as a sustained release or delayedrelease carrier. In such embodiments, the carrier can be any materialcapable of sustained or delayed release of the leptin peptide activeingredient to provide a more efficient administration, resulting in lessfrequent and/or decreased dosage of the active ingredient, ease ofhandling, and extended or delayed effects. Non-limiting examples of suchcarriers include liposomes, microsponges, microspheres, or microcapsulesof natural and synthetic polymers and the like. Liposomes may be formedfrom a variety of phospholipids such as cholesterol, stearylamines orphosphatidylcholines.

In some embodiments, the compositions of the present invention canfurther include one or more compatible active ingredients aimed atproviding the composition with another pharmaceutical effect in additionto that provided by a leptin composition, therapeutically active leptin,leptin mimic peptide or a derivative thereof “Compatible” as used hereinmeans that the active ingredients of such a composition are capable ofbeing combined with each other in such a manner so that there is nointeraction that would substantially reduce the efficacy of each activeingredient or the composition under ordinary use conditions. In anotheraspect of the present invention, the composition also may beadministered serially or in combination with other compositions fortreating diseases, conditions or disorders resulting from accumulationof amyloid peptides. For example, without limitation, such othercompositions may include monoclonal antibodies (such as monoclonalanti-β-Amyloids and monoclonal anti-β-secretases); and anti-inflammatorycompounds (including, but not limited to nonsteroidal anti-inflammatorydrugs (NSAIDs), such as ibuprofen, indomethacin, and flurbiprofen).Anti-inflammatory compounds have been shown to direct Aβ-loweringproperties in cell cultures as well as in transgenic models of AD-likeamyloidosis.

The concentration of the active substance is selected so as to exert itstherapeutic effect, but low enough to avoid significant side effectswithin the scope and sound judgment of the skilled artisan. Theeffective amount of the composition may vary with the age and physicalcondition of the biological subject being treated, the severity of thecondition, the duration of the treatment, the nature of concurrenttherapy, the specific compound, composition or other active ingredientemployed, the particular carrier utilized, and like factors. Those ofskill in the art can readily evaluate such factors and, based on thisinformation, determine the particular effective concentration of acomposition of the present invention to be used for an intended purpose.Additionally, in therapeutic applications of the present invention,compositions or medicants are administered to a patient suspected of,having, or already suffering from, such a disease, disorder or conditionin an amount sufficient to cure, or at least partially arrest, thesymptoms of the disease, disorder or condition, including itscomplications and intermediate pathological phenotypes in development ofthe disease, disorder or condition. In some methods, administration ofthe composition of the present invention reduces or eliminates cognitiveimpairment in patients that have not yet developed characteristicpathology of the disease, disorder or condition.

An amount adequate to accomplish therapeutic or prophylactic treatmentis defined herein as a therapeutically-effective dose. In bothprophylactic and therapeutic regimes, an amount of the compositions ofthe present invention is usually administered in several dosages until asufficient beneficial response has been achieved. Typically, theresponse is monitored and repeated dosages are given if the responsestarts to wane. A skilled artisan can determine a pharmaceuticallyeffective amount of the inventive compositions by determining the dosein a dosage unit (meaning unit of use) that elicits a given intensity ofeffect, hereinafter referred to as the “unit dose.” The term“dose-intensity relationship” refers to the manner in which theintensity of effect in an individual recipient relates to dose. Theintensity of effect generally designated is 50% of maximum intensity.The corresponding dose is called the 50% effective dose or individualED50. The use of the term “individual” distinguishes the ED50 based onthe intensity of effect as used herein from the median effective dose,also abbreviated ED50, determined from frequency of response data in apopulation. “Efficacy” as used herein refers to the property of thecompositions of the present invention to achieve the desired response,and “maximum efficacy” refers to the maximum achievable effect. Theamount of compounds in the compositions of the present invention whichwill be effective in the treatment of a particular disorder or conditionwill depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques. (See, for example, Goodmanand Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Harman,Lee E. Limbird, Eds.; McGraw Hill, New York, 2001; THE PHYSICIAN′S DESKREFERENCE, Medical Economics Company, Inc., Oradell, N.J., 1995; andDRUG FACTS AND COMPARISONS, FACTS AND COMPARISONS, INC., St. Louis, Mo.,1993). The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Variousadministration patterns will be apparent to those skilled in the art.

The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desiredtherapeutic effect. Preferably, the therapeutically effective amount ofthe compositions of the present invention is administered one or moretimes per day on a regular basis. A typical dose administered to asubject is between about 0.01 mg of the composition per kg (of bodyweight) per day and about 0.5 mg of the composition per kg (of bodyweight) per day. For example, without limitation, the minimum dose ofthe composition is contemplated as about 0.01 mg/kg/day, about 0.025mg/kg/day, about 0.05 mg/kg/day, about 0.075 mg/kg/day, about 0.08mg/kg/day, about 0.1 mg/kg/day, about 0.125 mg/kg/day, about 0.15mg/kg/day, about 0.175 mg/kg/day, about 0.2 mg/kg/day, about 0.225mg/kg/day, about 0.25 mg/kg/day, about 0.275 mg/kg/day, about 0.3mg/kg/day, about 0.325 mg/kg/day, about 0.35 mg/kg/day, about 0.375mg/kg/day, about 0.4 mg/kg/day, about 0.45 mg/kg/day, about 0.475mg/kg/day, or about 0.5 mg/kg/day and the maximum dose is contemplatedas about 0.5 mg/kg/day, about 0.475 mg/kg/day, about 0.45 mg/kg/day,about 0.4 mg/kg/day, about 0.375 mg/kg/day, about 0.35 mg/kg/day, about0.325 mg/kg/day, about 0.3 mg/kg/day, about 0.275 mg/kg/day, about 0.25mg/kg/day, bout 0.225 mg/kg/day, about 0.2 mg/kg/day, about 0.175mg/kg/day, about 0.15 mg/kg/day, about 0.125 mg/kg/day, about 0.1mg/kg/day, about 0.08 mg/kg/day, about 0.075 mg/kg/day, about 0.05mg/kg/day, about 0.025 mg/kg/day, or about 0.01 mg/kg/day. In someembodiments of the invention in humans, the dose may be about 0.01 mg toabout 0.3 mg of the composition per kg (of body weight) per day, and inother embodiments in humans, between 0.01 and 0.08 mg of the compositionper kg (of body weight) per day.

Additional compositions of the present invention can be prepared readilyusing technology is known in the art, such as that which is described inRemington's Pharmaceutical Sciences, 18th or 19th editions, published bythe Mack Publishing Company of Easton, Pa., which is incorporated hereinby reference.

Administration

According to another embodiment of the method, the method comprises thestep of implanting surgically or injecting a leptin composition gel,leptin composition slow-release solid or leptin composition semisolidinto the patient to deliver drug substance at the site of interest.Because the leptin composition gel, leptin composition slow-releasesolid or leptin composition semisolid agent is delivered specifically(locally) to the site, the dosage required to treat the progressivecognitive disorder will be appropriate, to reduce, prevent or circumventthe main side effect that prevents the administration of higher systemicdoses, e.g., toxicity. It is desired to deliver efficacious amounts ofthis agent to a specific site (without unwanted side effects).

Controlled Release Systems

The therapeutic agent(s), including, but not limited to, a leptincomposition, may be contained in controlled release systems. In order toprolong the effect of a drug, it often is desirable to slow theabsorption of the drug from subcutaneous, intrathecal, or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.

The term “controlled release” is intended to refer to anydrug-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. This refers to immediate aswell as non-immediate release formulations, with non-immediate releaseformulations including, but not limited to, sustained release anddelayed release formulations. The term “sustained release” (alsoreferred to as “extended release”) is used herein in its conventionalsense to refer to a drug formulation that provides for gradual releaseof a drug over an extended period of time, and that preferably, althoughnot necessarily, results in substantially constant blood levels of adrug over an extended time period. Alternatively, delayed absorption ofa parenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. The term “delayed release” isused herein in its conventional sense to refer to a drug formulation inwhich there is a time delay between administration of the formulationand the release of the drug there from. “Delayed release” may or may notinvolve gradual release of drug over an extended period of time, andthus may or may not be “sustained release.”

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. The term “long-term”release, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 7 days, and preferably about 30 to about 60 days. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

According to another embodiment, the pharmaceutically acceptable carrierof the present invention includes a sustained release or delayed releasecarrier. The carrier can be any material capable of sustained or delayedrelease of the compound to provide a more efficient administrationresulting in less frequent and/or decreased dosage of the compound, easeof handling, and extended or delayed effects on epithelial-relatedconditions.

According to another aspect, the described invention provides a methodof improving resilience of cognitive function in a subject in needthereof, the method comprising the step of (a) administering to thesubject a composition comprising: (i) a cognitive function-enhancingamount of a leptin composition, and (ii) a pharmaceutically acceptablecarrier. According to one embodiment, the leptin composition comprisesat least one of a leptin, a leptin mimic, a leptin derivative, anAMP-dependent protein kinase activator, a leptin agonist, a leptinblocker, a mimic of a leptin blocker, a leptin antagonist, anAMP-dependent protein kinase inhibitor; or pharmaceutically acceptablesalts thereof. According to another embodiment, the leptin compositionfurther comprises a second therapeutic agent. According to anotherembodiment, the second therapeutic agent is an antibiotic. According toanother embodiment, the second therapeutic agent is an anti-fungalagent. According to another embodiment, the second therapeutic agent isan anti-viral agent. According to another embodiment, the secondtherapeutic agent is an anti-protozoal agent. According to anotherembodiment, the second therapeutic agent is a steroidalanti-inflammatory agent. According to another embodiment, the secondtherapeutic agent is a non-steroidal anti-inflammatory agent. Accordingto another embodiment, the second therapeutic agent is an anti-oxidant.According to another embodiment, the second therapeutic agent is ahormone. According to another embodiment, the second therapeutic agentis a vitamin. According to another embodiment, the second therapeuticagent is an antihistamine agent. According to another embodiment, thesecond therapeutic agent is a chemotherapetic agent.

Those skilled in the art will recognize that initial indications of theappropriate therapeutic dosage of the compositions of the invention canbe determined in in vitro and in vivo animal model systems, and in humanclinical trials. One of skill in the art would know to use animalstudies and human experience to identify a dosage that can safely beadministered without generating toxicity or other side effects. Foracute treatment, it is preferred that the therapeutic dosage be close tothe maximum tolerated dose. For chronic preventive use, lower dosagesmay be desirable because of concerns about long term effects.

The effectiveness of the compositions and methods of the presentinvention can be assayed by a variety of protocols. The effects ofincreasing cognitive function in a human subject can be determined bymethods routine to those skilled in the art including, but not limitedto, both paper and pencil, and computer tests. One of skill in the artcan also directly measure amyloid peptide accumulation levels,neurofibrillary tangle formation and neurodegeneration in animal models.Furthermore, amyloid peptide may be measured in a sample of a subject'scerebrospinal fluid (C SF) obtained by spinal tap. One measure ofaccumulation of an amyloid peptide is an increase in levels circulatingin the blood of a subject. Such levels may be measured by SandwichEnzyme-linked-Immunoabsorbent-Assays (ELISAs), using a pair ofantibodies, one for capture and the other for detection. These methodsare well known by those of ordinary skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein also can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Where a range of values is provided, it isunderstood that each intervening value, to the tenth of the unit of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range and any other stated or interveningvalue in that stated range is encompassed within the invention. Theupper and lower limits of these smaller ranges which may independentlybe included in the smaller ranges is also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either both of those included limits are also included in theinvention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Reagents and Antibodies

Minimum essential medium (MEM) was purchased from ATCC (Manassas, Va.).Neurobasal medium, B27 supplement and L-glutamine were purchased fromGibco (Carlsbad, Calif.). Trypsin-EDTA andpenicillin-streptomycin-amphotercin solution were purchased from MPBiomedicals (Solon, Ohio). Fetal bovine serum (FBS), all-trans retinoicacid (RA), also known as ATRA, human recombinant leptin and humanrecombinant insulin were purchased from Sigma-Aldrich (St. Louis, Mo.).5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR), a drug widelyused to activate AMP-dependent protein kinase (AMPK) experimentally, waspurchased from Cell Signaling Technology (Danvers, Mass.). Uponactivation, AMPK is known to promote lipolysis and to inhibitlipogenesis.

Rabbit anti-AMPKα (pThr¹⁷²), Rabbit anti-AMPKα (total) and tau (pSer³⁹⁶)mouse mAb were purchased from Cell Signaling Technology. Tau mouse mAb(clone 5E2) for detection of total tau was purchased from Upstate CellSignaling Solutions (Lake Placid, N.Y.). PHF-tau mouse mAb (clone AT8)was purchased from Pierce Biotechnology (Rockford, Ill.). PHF-1 mousemAb was a gift from Dr. Peter Davies, Albert Einstein College ofMedicine (Bronx, N.Y.). Rabbit anti-leptin receptor (OB-R) and α-tubulinmouse mAb were purchased from Affinity BioReagents (Golden, Colo.).Insulin receptor (β-subunit) mAb was purchased from Millipore(Billerica, Mass.).

Culture of Cell Lines

The human neuroblastoma, SH-SY5Y, and embryonal carcinoma, NTera-2(NT2), cell lines were purchased from American Type Culture Collection(ATCC). Cell culture was performed according to manufacturer's specificguidelines. Briefly, SY5Y and NT2 cells were propagated on 25 cm²tissue-culture flasks (Corning; Corning, N.Y.) in minimum essentialmedium (MEM) (Eagle) containing 10% fetal bovine serum (FBS) until80-90% confluence was established. SY5Y and NT2 cells were detached fromthe flask by 0.1% trypsin-EDTA and gentle scraping, respectively, andsub-cultured at a ratio of 1:5.

Neuronal Induction

To induce neuronal differentiation, 1×10⁶ SY5Y or NT2 cells were seededin 25 or 75 cm² tissue-culture flasks, respectively. Cells were grown inneuronal induction medium (NIM), which consisted of MEM containing 5%FBS supplemented with 10 μM RA. SY5Y were grown in NIM for 6 days, andswitched to serum-free NIM prior to treatment and harvesting on day 7.To induce neuronal differentiation of NT2 cells was based on apreviously described protocol [P. W. Andrews, Retinoic acid inducesneuronal differentiation of a cloned human embryonal carcinoma cell linein vitro, Dev. Biol. 103 (1984) 285-293, which is incorporated herein byreference]. Briefly, NT2 cells were cultured in NIM for 5 weeks, with50% NIM replacement every 3 days. Differentiated NT2 cells (NT2N) wereswitched to serum-free NIM on the day prior to treatment and harvesting.

Culture of Rat Primary Neurons

Primary rat cortical neurons were purchased from BrainBits LLC(Sprinfield, Ill.), and cultured as per manufacturer's instructions.Briefly, tissues were dispersed and supernatant was transferred to a newtube and centrifuged for 1 min at 1100 rpm. The neurons then were seededin 6-well plates coated with poly-D-lysine (BD Biosciences; San Jose,Calif.) and grown in Neurobasal medium supplemented with B27 supplement(Invitrogen) and 0.5 mM L-glutamine. Medium was changed after 4 days,and at 7 days in culture the neurons were treated and harvested.

Protein Extraction and Western Blotting

Western blot (or immunoblot) analysis is a method to detect a specificprotein in a given sample of a tissue homogenate or extract generallyuses SDS-gel electrophoresis to separate typically denatured proteins bythe molecular weight of the polypeptide. Proteins are then transferredto a membrane (typically nitrocellulose or PVDF) where they are detectedusing antibodies specific to the target protein.

Following treatment with leptin, insulin and/or AICAR, SY5Y, NT2N andrat cortical neurons were harvested by scraping. Cell pellets werewashed twice in ice-cold 1×PBS (phosphate buffered saline) (pH 7.4),resuspended in protease and phosphotase inhibitor-supplemented 1×RIPAlysis/extraction buffer consisting of 25 mM Tris-HCl, pH 7.6, 150 mMNaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS (Pierce), and thensubjected to freeze/thaw cycles in a dry ice/ethanol bath. Cell-free,whole cell lysates were obtained and total protein was determined withthe Coomassie (Bradford) Protein Assay Kit (Pierce). Whole cell extracts(25 μg) were analyzed by western blots using 10% SDS-PAGE pre-cast gels(Lonza; Rockland, Me.), and the separated proteins were transferred ontopolyvinylidene difluoride membranes (Millipore). Membranes wereincubated overnight at 4° C. with primary antibodies and then detectedthe following day by 2 hr incubation with HRP-conjugated IgG. Allprimary antibodies, except tau-pSer³⁹⁶ (1:500), total tau (1:500) andPHF-tau AT8 (1:200), and secondary antibodies were used at finaldilutions of 1:1,000 and 1:10,000, respectively. HRP was developed withSuperSignal West Pico Chemiluminescent Substrate (Pierce), and imagedusing a BioRad (Hercules, Calif.) ChemiDoc XRS System. The membraneswere stripped with Restore PLUS Western Blot Stripping Buffer (Pierce)for reprobing with other antibodies. Blocking buffer consisted of 5%milk in 0.1% Tween in TBS (Tris buffered saline).

Statistical Analysis

Statistical data analyses were performed with analysis of variance andTukey-Kramer multiple comparisons test. Densitometric analyses wereperformed using the UN-SCAN-IT gel 6.1 software (Silk Scientific; Orem,Utah). p<0.05 was considered statistically significant.

Example 1 Leptin and Tau Phosphorylation in RA-Induced SY5Y Cells

RA induction of the human neuroblastoma cell line, SY5Y, has beenreported to induce hyperphosphorylation of tau at AD-related sites. Wetherefore utilized SY5Y cells induced with retinoic acid (RA-SY5Y) for 7days as our primary in vitro model to investigate the effects of leptinand other treatments on tau phosphorylation.

The first set of studies examined expression of the leptin receptor(OB-R) in RA-SY5Y cells treated with 400 ng/ml leptin or placebo. Bothtreated and placebo cells were found to express relatively high levelsof OB-R (FIG. 1A). We next determined whether leptin had an effect ontau phosphorylation. Cells were treated for a range of time periods with400 ng/ml leptin or placebo, and phosphorylation of tau at Ser³⁹⁶, asite within the microtubule-binding region of tau, was measured (FIG. 1Band FIG. 1C). Significant (p<0.05) decreases in tau (Ser³⁹⁶)phosphorylation were observed in cells treated with leptin for 1 hour, 2hours or 4 hours compared to placebo (FIG. 1C; far right bars). Nochange in tau (Ser³⁹⁶) phosphorylation was observed in cells treatedwith leptin for 24 hours compared to 4 hours (data not shown).

To determine the dose-response relationship between leptin and tauSer³⁹⁶ phosphorylation, RA-SY5Y cells were treated with leptin for 4hours at a range of concentrations (FIG. 1D and FIG. 1E). We observed asignificant (p<0.05) decrease in tau (Ser³⁹⁶) phosphorylation in cellstreated with 100 ng/ml leptin (FIG. 1E; second bar from left).Decreasing tau (Ser³⁹⁶) phosphorylation was observed up to aconcentration of 1600 ng/ml leptin (second bar from right), whichproduced the maximal effect. Estimation of the 50% inhibitoryconcentration (IC₅₀) of leptin for tau (Ser³⁹⁶) phosphorylation provideda value of 750 ng/ml, or 46.9 nM.

Example 2 Insulin and Tau Phosphorylation in RA-Induced SY5Y Cells

We tested the effect of insulin treatment on tau (Ser³⁹⁶)phosphorylation in RA-SY5Y cells and compared it to that of leptin.

The first set of studies examined expression of the insulin receptor inRA-SY5Y cells treated with 10 μM insulin or placebo. Both insulin andplacebo-treated cells were found to express high levels of insulinreceptor (FIG. 2A). We next determined the effect of insulin on tauphosphorylation. Cells were treated for a range of time periods with 10μM insulin or placebo, and phosphorylation of tau (Ser³⁹⁶) was measured(FIG. 2B and FIG. 2C). Significant (p<0.05) decreases in tau (Ser³⁹⁶)phosphorylation were observed in cells treated with insulin for 2 hoursor 4 hours compared to placebo-treated cells (FIG. 2C; far right bars).No change in tau (Ser³⁹⁶) phosphorylation was observed in cells treatedwith insulin for 24 hours compared to 4 hours (data not shown).

As in the leptin studies (FIG. 1D and FIG. 1E), a dose-response curvefor insulin on tau (Ser³⁹⁶) phosphorylation was established in RA-SY5Ycells (FIG. 2D and FIG. 2E). We observed a significant (p<0.05) decreasein tau (Ser³⁹⁶) phosphorylation in cells treated with 10 μM insulin(FIG. 2E; third bar from right). Further, maximum decrease of tau(Ser³⁹⁶) phosphorylation was observed at a concentration of 20 μMinsulin (second bar from right). Estimation of the 50% inhibitoryconcentration (IC₅₀) of insulin for tau (Ser³⁹⁶) phosphorylationprovided a value of 13.8 μM.

Summary

The effect of leptin on the level of tau phosphorylation at sites knownto be hyperphosphorylated in AD was studied. RA-induced, human SY5Yexpress hyperphosphorylated tau, and thus were utilized in our treatmentmodel. Since insulin reduces the level of phosphorylated tau in both invitro and in vivo models, our studies began by comparing the efficacy ofleptin to insulin (FIGS. 1 and 2). Leptin was found to reduce tauphosphorylation by 50% at a concentration (FIG. 1; IC₅₀=46.9 nM) thatwas 300-fold less than that of insulin (FIG. 2; IC₅₀=13.8 μM).

Example 3 Combined Leptin and Insulin Treatment and Tau Phosphorylation

RA-SY5Y cells were treated for 4 hours with sub-optimal or maximumeffect doses, either in combination or alone, of leptin and/or insulin,and tau (Ser³⁹⁶) phosphorylation was measured (FIG. 3A and FIG. 3B). Asignificant (p<0.05) decrease in phosphorylation was observed in cellstreated with sub-optimal combinations of leptin (100 ng/ml) and insulin(1 μM) compared to either treatment alone (FIG. 3B; first, third andfifth bars from left). Co-treatment with maximum effect doses of leptin(1600 ng/ml) and insulin (20 μM) produced the most significant (p<0.01)decrease in phosphorylation (first bar from right) compared toplacebo-treated. Co-treatment with maximum effect doses of leptin andinsulin did not produce a significant (p>0.05) reduction in tau (Ser³⁹⁶)phosphorylation compared to either treatment alone.

Summary

The combined treatment with sub-optimal doses of leptin (100 ng/ml) andinsulin (1 μM) produced a significant decrease in tau phosphorylationcompared to either treatment alone (FIG. 3). This result demonstratesthe potential benefits of a combinatorial treatment for AD, as leptinand insulin may produce an additive effect.

Example 4 Reversibility of Leptin- and Insulin-Induced Dephosphorylation

Tau phosphorylation has been reported to increase with cold temperaturestress in animals. We thus utilized a similar approach to determinewhether the leptin- and insulin-induced dephosphorylation of tau atSer³⁹⁶ was reversible. RA-SY5Y were co-treated with leptin (1600 ng/ml)and insulin (20 μM) for 4 hours, or placebo. At the end of the treatmentperiod, cells were either harvested or post-treated with ice-cold PBS(pH 7.4) for 10 minutes or 1 hour (FIG. 3C and FIG. 3D). Cellspost-treated with cold PBS for 10 minutes showed a significant (p<0.05)increase in tau phosphorylation compared to co-treatment alone (FIG. 3D;first and second bars from left). Cells post-treated with cold PBS for 1hour showed significant (p<0.01) hyperphosphorylation of tau compared tocells with no treatment at all (first bar from right). These resultssuggest that the effects of leptin and insulin on dephosphorylation oftau are reversible. The results also demonstrate antibody specificityregarding the phosphorylated form of tau.

Example 5.1 Leptin, Insulin and Tau Phosphorylation at Other AD-RelatedSites

To evaluate if the observed effects of leptin and insulin on tauphosphorylation at Ser³⁹⁶ (FIG. 1 and FIG. 2) is consistent with otherAD-related sites, antibodies raised against tau epitopes known to bephosphorylated in paired helical filament (PHF) tau were utilized. PHFsare a principal component of NFT pathology, which results from tauhyperphosphorylation and subsequent microtubule destabilization andoligomer formation. Tau phosphorylated at Ser^(396/404) andSer²⁰²/Thr²⁰⁵ is recognized by PHF-1 (mouse) and AT8 (mouse) antibodies,respectively.

RA-SY5Y cells were treated with leptin and/or insulin as in FIG. 3A andFIG. 3B, and phosphorylation of specific tau sites was measured (Table1).

TABLE 1 Relative tau phosphorylation in treated neuronal culturesTreatment Leptin Leptin 100 ng/ml + 1600 ng/ml + Cell Phospho- Non-Leptin Leptin Leptin Insulin Insulin Insulin Insulin Type Site Treated100 ng/ml 800 ng/ml 1600 ng/ml 1 μM 20 μM 1 μM 20 μM RA- pSer³⁹⁶ 0 −26 ±6* ND −51 ± 5* −23 ± 4* −47 ± 14* −58 ± 10* −69 ± 12* SY5Y PHF-1 0 −20 ±19 ND −67 ± 4* −37 ± 11* −80 ± 7* −72 ± 3* −84 ± 6* AT8 0 −10 ± 5 ND −60± 19* −40 ± 13* −57 ± 14* −61 ± 17* −66 ± 21* NT2N pSer³⁹⁶ 0 −27 ± 6* ND−27 ± 5* −23 ± 6* −53 ± 10* −42 ± 7* −48 ± 1* Rat 1° PHF-1 0 ND −75 ±18* ND ND ND ND ND Neuron AT8 0 ND  5 ± 23 ND ND ND ND ND

Briefly, RA-induced SY5Y and NT2N were treated with low and highconcentrations of leptin (100 ng/ml or 1600 ng/ml) and/or insulin (1 μMor 20 μM) for 4 hours, or non-treated (placebo). Primary rat corticalneurons were treated with leptin for 24 hours or placebo. Whole cellextracts were prepared and analyzed by Western blot with phosphorylatedtau-specific antibodies (pSer³⁹⁶, PHF-1 or AT8). Membranes were strippedand re-probed with anti-tau (total) for normalization. Normalized banddensities were analyzed by densitometry and results are presented as themean±SD percent fold change, relative to non-treated samples, which werearbitrarily assigned a value of 0. (ND—Not Determined). (*p<0.05 vsnon-treated).

Leptin and/or insulin treatment was observed to have a similar effect onthe phosphorylation of tau as detected by PHF-1 and AT8 antibodies(Table 1). The only observable difference was that leptin at 100 ng/mlwas unable to induce a significant (p>0.05) decrease in phosphorylationof tau, compared to that observed with pSer³⁹⁶ antibody. These findingsdemonstrate that both leptin and insulin treatment of RA-SY5Y cellsreduces phosphorylation of at least two separate AD-related tau sites.

Example 5.2 Leptin, Insulin and Tau Phosphorylation in Other NeuronalCells

We next determined whether the effect of leptin and/or insulin on tauphosphorylation was unique to RA-SY5Y cells or consistent with otherneuronal cells. For this approach, we utilized human NT2 cells, whichundergo neuronal differentiation with R^(A) treatment (NT2N), as well asrat primary cortical neurons.

NT2N cells were treated with leptin and/or insulin as in FIG. 3A andFIG. 3B, and tau phosphorylation at Ser³⁹⁶ was measured (Table 1).Insulin and combined insulin/leptin treatment were observed to have asimilar effect to that observed with RA-SY5Y cells (Table 1).

For the rat primary neurons, we determined the effect of 24 hour leptintreatment on phosphorylation of tau, as detected by PHF-1 and AT8antibodies (Table 1). A mid-range dose of leptin (800 ng/ml) was chosen,since this concentration produced a 50% decrease (ID₅₀) in tauphosphorylation within RA-SY5Y (FIG. 1). Leptin produced a significant(p<0.05) decrease in tau phosphorylation, as detected by PHF-1 antibodycompared to placebo-treated cells (Table 1). However, the leptin-induceddecrease in tau phosphorylation was not detected by the AT8 antibody(Table 1).

In summary, leptin induces a reduction in phosphorylation of tau atSer^(396/404) (as detected by PHF-1 antibody) in several neuronal cells.Further, it induces a reduction of tau phosphorylation at Ser²⁰²/Thr²⁰⁵(as detected by AT8 antibody) in most but not all neuronal cell typestested.

Summary

Tau phosphorylation in human NT2N cells and rat primary cortical neurons(Table 1) was examined to demonstrate the effects of leptin wereconsistent with other neuronal systems. Similar results were observed asin RA-SY5Y except that leptin did not significantly changephosphorylation of Ser²⁰²/Thr²⁰⁵ (AT8 mouse mAb) in rat corticalneurons. Without being limited by theory, this result may be related tothe antibodies' species specificity.

Example 6 AMPK Signaling and Tau Phosphorylation in RA-SY5Y Cells

The energy homeostasis enzyme AMP-activated protein kinase (AMPK) wasdirectly stimulated with the cell-permeable activator, AICAR to studythe influence of leptin and insulin in modulating tau phosphorylation.

AICAR treatment produced a large increase in pThr¹⁷² AMPKα band density(FIG. 4A, top row), thus demonstrating efficient activation of AMPKα. Wenext determined the effect of AICAR on tau phosphorylation. RA-SY5Y weretreated for various amounts of time with 1 mM AICAR or placebo-treated(FIG. 4B and FIG. 4C). Significant (p<0.05) decreases in Ser³⁹⁶phosphorylation were observed in cells treated with AICAR from 10minutes to 4 hours, compared to placebo (FIG. 4C; gray bars).

RA-SY5Y were treated with AICAR for 1 hour at a range of concentrations,to establish a dose-response relationship (FIG. 4D and FIG. 4E). Weobserved a significant (p<0.05) decrease in Ser³⁹⁶ phosphorylation incells treated with 1 mM AICAR (FIG. 4E; third bar from right).Decreasing Ser³⁹⁶ phosphorylation was observed up to a concentration of2 mM AICAR (second bar from right), which produced the maximal effect.Estimation of the 50% inhibitory concentration (IC₅₀) of AICAR for tau(Ser³⁹⁶) phosphorylation provided a value of 2.7 mM.

In summary, the observed results suggest that activation of AMPKα, byeither leptin or insulin, could produce similar effects on tauphosphorylation at AD-related sites.

The point of convergence of the post-receptor signaling pathways in tauphosphorylation, was investigated. The energy homeostasis enzyme AMPK(FIG. 4) is known to be activated by insulin and leptin and is alsoknown to interact with glycogen synthase kinase-3β (GSK-3β). Activationof AMPK with AICAR produced significant changes in tau phosphorylationwithin 10 minutes. These findings suggest that AMPK may provide a noveltherapeutic target for reducing AD-related tau phosphorylation. Wedemonstrated that activation of AMPK mimics the leptin/insulin effect.

Example 7 Clinical Trials

The clinical development of leptin in humans is investigated. A pilottrial, [placebo-cotrolled double blinded] involving three groups ofequal number of patients, diagnosed with early-stage Alzheimer'sdisease, receive by subcutaneous injections 0 mg (placebo), 5 mg, or 10mg of leptin once daily for 16 weeks. CSF and serum samples are obtainedin the beginning, during and at the end of the trial and Ab40, Ab42 andphosphor-tau are measured. Patients also receive neuropsychologicalevaluations at the beginning and at the end of the trial. This trialvalidates the preclinical findings and demonstrates leptin's value inselectively targeting both pathologies of AD.

The clinical trial data, taken with the preclinical data demonstratesthat leptin ameliorates both Aβ and tau-related pathologies. Togetherwith leptin's pharmacological profile these data support its use as anovel therapeutic for Alzheimer's disease.

Example 8 RA-Induced SY5Y and NT2N Treated with Leptin or Insulin

RA-induced SY5Y and NT2N were treated with low or high concentrations ofleptin (100 ng/ml or 1600 ng/ml, respectively) and/or insulin (1 μM or20 μm, respectively) for 4 hours, or non-treated (placebo) (Table 1).Primary rat cortical neurons were treated with leptin for 24 hours orplacebo. Whole cell extracts were prepared and analyzed by western blotwith phosphorylated tau-specific antibodies (pSer³⁹⁶, PHF-1 or AT8).Membranes were stripped and re-probed with anti-tau (total) fornormalization. Normalized band densities were analyzed by densitometryand results are presented as the mean±SD percent fold change, relativeto non-treated samples, which were arbitrarily assigned a value of 0.(ND—Not Determined). *p<0.05 vs. non-treated.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for treating a progressive cognitivedisorder, the method comprising the step of: (a) administering to asubject in need thereof a first composition comprising (i) aphosphorylated tau accumulation-modulating amount of a leptincomposition, or a pharmaceutically acceptable salt thereof, and (ii) apharmaceutically acceptable carrier, and (b) modulating accumulation ofphosphorylated tau in cerebrospinal fluid of the subject.
 2. The methodaccording to claim 1, wherein the progressive cognitive disorder isselected from the group consisting of progressive supranuclear palsy,dementia, dementia pugilistica, Creutzfeldt-Jakob disease,frontotemporal dementia, Pick's disease, and FTDP-17 (parkinsonism)corticobasal degeneration.
 3. The method according to claim 1, whereinthe leptin composition is a leptin, or a pharmaceutically acceptablesalt thereof.
 4. The method according to claim 1, wherein the leptincomposition is a leptin mimic, or a pharmaceutically acceptable saltthereof.
 5. The method according to claim 1, wherein the leptincomposition is a leptin derivative, or a pharmaceutically acceptablesalt thereof.
 6. The method according to claim 1, wherein the leptincomposition is a leptin agonist, or a pharmaceutically acceptable saltthereof.
 7. The method according to claim 1, wherein the phosphorylatedtau accumulation modulating amount is an amount from about 0.01 mg/kgbody weight to about 100 mg/kg body weight.
 8. The method according toclaim 1, wherein the first composition further comprises a secondtherapeutic agent.
 9. The method according to claim 8, wherein thesecond therapeutic agent is at least one of an antibiotic, ananti-fungal agent, an antiviral agent, an anti-protozoal agent, asteroidal anti-inflammatory agent, a non-steroidal anti-inflammatoryagent, an anti-oxidant; a hormone; a vitamin; an antihistamine agent.and a chemotherapeutic agent.
 10. The method according to claim 1,wherein the progressive disorder comprises accumulation ofneurofibrillary tangles in brain.
 11. A method for improving resilienceof cognitive function in a subject in need thereof, the methodcomprising the step of (a) administering to the subject a compositioncomprising: i. a cognitive function-enhancing amount of a leptincomposition, and ii. a pharmaceutically acceptable carrier; and (b)modulating accumulation of phosphorylated tau in cerebrospinal fluid ofthe subject.
 12. The method according to claim 11, wherein the leptincomposition comprises at least one of a leptin, a leptin mimic, a leptinderivative, an AMP-dependent protein kinase activator, a leptin agonist,a leptin blocker, a mimic of a leptin blocker, a leptin antagonist, anAMP-dependent protein kinase inhibitor; or pharmaceutically acceptablesalts thereof.
 13. The method according to claim 11, wherein the leptincomposition further comprises a second therapeutic agent.
 14. The methodaccording to claim 13, wherein the second therapeutic agent is at leastone of an antibiotic, an anti-fungal agent, an antiviral agent, ananti-protozoal agent, a steroidal anti-inflammatory agent, anon-steroidal anti-inflammatory agent, an anti-oxidant; a hormone; avitamin; an antihistamine agent. and a chemotherapeutic agent.